The present invention relates generally to chemoattractant cytokines, called chemokines, and more specifically to truncated or variant forms of chemokines which have functions different from their wild-type counterparts, methods of use and methods of producing such variant chemokines.
Immunomodulatory proteins include chemotactic cytokines, called xe2x80x9cchemokinesxe2x80x9d. Chemokines are small molecular weight immune ligands which are chemoattractants for leukocytes, such as especially neutrophils, basophils, monocytes and T cells. There are two major classes of chemokines which both contain four conserved cysteine residues which form disulfide bonds in the tertiary structure of the proteins. The xcex1 class is designated C-X-C (where X is any amilno acid), which includes IL-8, CTAP-III, gro/MGSA and ENA-78; and the xcex2 class, designated C-C, which includes MCP-1, MIP-1xcex1 and xcex2, and regulated on activation, normal T expressed and secreted protein (RANTES). The designations of the classes are according to whether an intervening residue spaces the first two cysteines in the motif. In general, most C-X-C chemokines are chemoattractants for neutrophils but not monocytes, whereas C-C chemokines appear to attract monocytes but not neutrophils. Recently, a third group of chemokines, the xe2x80x9cCxe2x80x9d group, was designated by the discovery of a new protein called lymphotactin (Kelner, et al., Science, 266:1395-1933, 1994). The chemokine family is believed to be critically important in the infiltration of lymphocytes and monocytes into sites of inflammation.
Monocytes differentiate into macrophages as they migrate from the blood to tissues during immune surveillance. At sites of inflammation, monocyte infiltration and macrophage accumulation are coordinated, in part, by chemokines (1). The mechanisms that control the recruitment of monocytes and macrophages by chemoattractants have not been clearly defined, but they may include regulation of the expression of chemokines and their receptors (2) as well as the modification of chemokine activity by posttranslational processing (3-5). Several chemokines share a conserved NH2-X-Pro sequence (X, any amino acid) at the NH2-terminus (6), which conforms to the substrate specificity of dipeptidyl exopeptidase IV (DPPIV) (7). DPPIV cleaves the first two amino acids from peptides with penultimate proline or alanine residues, although no natural substrate with immune function has been identified. This enzyme is also a leukocyte differentiation antigen, known as CD26 (8-10), that is expressed on the cell surface mostly by T lymphocytes and macrophages. Expression of CD26 has been associated with T cell activation (8-10) and with susceptibility of a T cell line to infection with macrophage-tropic (M-tropic) HIV-1 (11).
The present invention is based on the discovery that chemokines having a particular N-terminal motif are natural substrates for a dipeptidyl dipeptidase (DPPIV). Prior to the present invention, it was known that CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV (DPPIV) activity but natural substrates had not been identified. The present invention shows that several chemokines, including RANTES (regulated on activation, normal T expressed and secreted) are substrates for recombinant soluble human CD26 (sCD26). The present invention shows that DPPIV, e.g., CD26-mediated processing, together with cell activation induces changes in receptor expression and provides a mechanism for differential cell recruitment and for the regulation of target cell specificity of chemokines.
Abbreviations: [Ca2+]i, cytosolic free Ca2+ concentration; DPPIV, dipeptidyl peptidase IV; ES-MS, electrospray mass spectromety; M-tropic, macrophage-tropic; pNA, p-nitroanilide; rh, recombinant human; sCD26, soluble CD26.
In a first embodiment, the invention provides the nucleotide and amino acid sequence of truncated RANTES (3-68), which is the same as the wild-type RANTES with a Serine/Proline truncation at positions 1 and 2 from the N-terminus, respectively.
In another embodiment, the invention provides a method for identifying a compound which modulates dipeptidyl peptidase IV (DPPIV)-mediated chemokine processing. The method includes a) incubating components comprising the compound, DPPIV and a chemokine under conditions sufficient to allow the components to interact; and b) determining the N-terminal amino acid sequence of the chemokine before and after incubating in the presence of the compound. Modulation of DPPIV-mediated chemokine processing may be inhibition or stimulation of processing, for example. Compounds which modulate such processing include peptides, peptidomimetics, and other small molecule compounds.
In another embodiment, the invention provides a method of inhibiting membrane fusion between HIV and a target cell or between an HIV-infected cell and a CD4 positive uninfected cell by contacting the target or CD4 positive cell with a fusion-inhibiting effective amount of the polypeptide of SEQ ID NO:2 (RANTES 3-68).
The invention also provides a method of treating a subject having or at risk of having an HIV infection or disorder, including administering to the subject, a therapeutically effective amount of a polypeptide of SEQ ID NO:2, wherein the polypeptide inhibits cell-cell fusion in cells infected with HIV The invention also provides a method of treating a subject having an HIV-related disorder associated with expression of CCR5 comprising administering to an HIV infected or susceptible cell of the subject, a polypeptide of SEQ ID NO:2 or a nucleic acid sequence encoding the polypeptide of SEQ ID NO:2 or other variant chemokine. Preferably, the subject is a human.
Also included are pharmaceutical compositions including the polypeptide of SEQ ID NO:2 or CD26, in pharmaceutically acceptable carriers.
In yet another embodiment, the invention provides a method for producing a variant chemokine having an activity different from the activity of the wild-type chemokine, including contacting the wild-type chemokine with an N-terminal processing effective amount of dipeptidyl peptidase IV (DPPIV), thereby truncating the chemokine and producing a variant chemokine. Chemokines may include, but are not limited to, RANTES, MIP-1, IP-10, eotaxin, MDC, and MCP-2.
The invention also provides a method for inhibiting HIV-1 replication in a host cell susceptible to HIV-1 infection, comprising contacting the cell or the host with an effective amount of dipeptidyl peptidase IV (DPPIV) enzyme such that macrophage-derived chemokine (MDC) is cleaved to produce truncated MDC, thereby providing antiviral activity and inhibiting HIV-1 replication and a A method for inhibiting HIV-1 replication in a host cell susceptible to HIV-1 infection, comprising contacting the cell or the host with an effective amount of dipeptidyl peptidase IV (DPPIV) enzyme such that RANTES is cleaved to produce truncated RANTES, thereby providing antiviral activity and inhibiting HIV-1 replication.
In another embodiment, the invention provides a method for inhibiting dipeptidyl peptidase IV (DPPIV)-mediated chemokine processing comprising contacting DPPIV with an inhibiting effective amount of a compound which inhibits DPPIV expression or activity.
In another embodiment, the invention provides a method for inhibiting an allergic or inflammatory reaction in a subject, comprising administering to the subject an effective amount of Dipeptidyl peptidase IV (DPPIV) enzyme such that a chemokine is cleaved to produce a truncated chemokine, thereby inhibiting an allergic or inflammatory reaction. Preferably, the chemokine is eotaxin.
In another embodiment, the invention provides a method for accelerating angiogenesis or wound healing in a subject, comprising administering to the subject an effective amount of an inhibitor of dipeptidyl peptidase IV (DPPIV) enzyme activity or gene expression or a DPPIV-insensitive chemokine, such that chemolcine processing is inhibited, thereby accelerating angiogenesis or wound healing. One exemplary chemokine useful in the method for accelerating angiogenesis is IP-10.
In all of the above methods, the exemplary DPPIV shown in the present invention is CD26.
In yet another embodiment, the invention provides a method for diagnosis or prognosis of a subject having a chemokine-associated disorder. The method includes identifying the presence of a chemokine of interest from a specimen isolated from the subject; determining the amino-terminal sequence of the chemokine, wherein a full-length amino acid sequence is indicative of the presence of a wild-type chemokine polypeptide and a truncated amino-terminal sequence is indicative of the presence of a variant chemokine; and determining the concentration of wild-type chemokine as compared to variant chemokine, thereby providing a diagnosis of the subject.